Anti-IL-33 antibodies and uses thereof for disease treatment

ABSTRACT

Antibodies which bind and neutralize human IL-33, and methods of using same, are provided, said antibodies are useful as agents for treating conditions associated with allergic disease including treating atopic dermatitis.

The present invention is in the field of medicine. More particularly,the present invention relates to antibodies directed againstinterleukin-33 (IL-33) and pharmaceutical compositions thereof. Theantibodies of the present invention are expected to be useful in thetreatment of atopic dermatitis.

Atopic dermatitis (also known as atopic eczema) is a chronicinflammatory allergic skin disease that is characterized by recurrentred, itchy lesions. Two major pathophysiological abnormalities areobserved in atopic dermatitis patients; cutaneous inflammation due toinappropriate immune responses in the skin and altered epidermalstructure and function. The inflammatory infiltrate is comprised of amixture of cells, in particular immune cells expressing IL-4, IL-5 andIL-13. These cytokines are often elevated in other allergic diseasesincluding asthma and allergic rhinitis.

Atopic dermatitis has been described as the leading non-fatal healthburden attributable to skin diseases. Although it most often starts ininfancy and affects two out of ten children, it is also highly prevalentin adults. Most adults who suffer from chronic atopic dermatitis havehad nearly lifelong disease. Itch, sleep deprivation, and socialembarrassment due to visible lesions have substantial effects on thepsychosocial wellbeing of patients and their relatives. In children, theeffect of atopic dermatitis on health-related quality of life is similarto that of other major childhood disorders, such as asthma and diabetes.Most people who have atopic dermatitis have a personal or family historyof allergies.

Currently, atopic dermatitis cannot be cured, and the aim of diseasemanagement is to control or improve symptoms and achieve long-termdisease control with a multistep approach. The main principles arecontinuous epidermal barrier repair with emollients, avoidance ofindividual trigger factors, and anti-inflammatory therapy with topicalcorticosteroids or calcineurin inhibitors. In severely affected cases,phototherapy or systemic immunosuppressants are indicated. Hence, thereis a need for additional efficacious therapies for patients with atopicdermatitis.

IL-33 is a member of the IL-1 cytokine superfamily, and is mainlyexpressed by keratinocytes, epithelial cells, and endothelial cells.IL-33 activates several types of innate and acquired immune cellscausing the production of other pro-inflammatory mediators, and it ismost frequently characterized as an epithelial cytokine that promotestype 2 or T helper 2 (Th2) immune responses. There are a number ofantibodies that bind to and neutralize IL-33 known in the art. Forexample, United States Publication No. 20160168242A1 and InternationalPublication No. WO2015/106080A2 disclose certain antibodies that bindhuman IL-33. However, there is a need for alternative antibodies thatbind human IL-33 with high affinity and are therapeutically effective intreating allergic diseases such as atopic dermatitis. Improved affinityand superior IC₅₀ may enable dosing benefits.

The present invention addresses the need for an alternative antibodytherapy for patients having allergic diseases such as atopic dermatitis,food allergy, allergic rhinitis, and asthma. In some embodiments, theallergic disease is atopic dermatitis. The present invention alsoaddresses the need for an alternative antibody therapy for patientshaving persistent inflammatory diseases including eosinophilicesophagitis, scleroderma/systemic sclerosis, ulcerative colitis, andchronic obstructive pulmonary disease. The present invention alsoaddresses the need for an alternative antibody therapy for patientshaving Crohn's disease.

The present invention provides antibodies that bind human IL-33. In someembodiments, the antibody comprises a light chain variable region (LCVR)and a heavy chain variable region (HCVR), wherein the LCVR comprisescomplementarity determining regions (CDRs) LCDR1, LCDR2, and LCDR3 andthe HCVR comprises CDRs HCDR1, HCDR2, and HCDR3, and wherein the aminoacid sequence of LCDR1 is SEQ ID NO: 16, the amino acid sequence ofLCDR2 is SEQ ID NO: 17, the amino acid sequence of LCDR3 is SEQ ID NO:18, the amino acid sequence of HCDR1 is SEQ ID NO: 13, the amino acidsequence of HCDR2 is SEQ ID NO: 14, and the amino acid sequence of HCDR3is SEQ ID NO: 15. In some such embodiments, Xaa at position 6 of SEQ IDNO: 13 is Ser, Xaa at position 2 of SEQ ID NO: 15 is Leu, Xaa atposition 6 of SEQ ID NO: 17 is ala, and Xaa at position 6 of SEQ ID NO:18 is Ser. In other such embodiments, Xaa at position 6 of SEQ ID NO: 13is Phe, Xaa at position 2 of SEQ ID NO: 15 is Leu, Xaa at position 6 ofSEQ ID NO: 17 is Leu, and Xaa at position 6 of SEQ ID NO: 18 is Pro. Inother such embodiments, Xaa at position 6 of SEQ ID NO: 13 is Phe, Xaaat position 2 of SEQ ID NO: 15 is Ile, Xaa at position 6 of SEQ ID NO:17 is Leu, and Xaa at position 6 of SEQ ID NO: 18 is Pro.

The present invention also provides an antibody that binds human IL-33,wherein the antibody comprises a LCVR and a HCVR, and wherein the aminoacid sequence of the LCVR is SEQ ID NO: 4, SEQ ID NO: 8, or SEQ ID NO:12, and the amino acid sequence of the HCVR is SEQ ID NO: 3, SEQ ID NO:7, or SEQ ID NO: 11. In a particular embodiment, the amino acid sequenceof the LCVR is SEQ ID NO: 4, and the amino acid sequence of the HCVR isSEQ ID NO: 3. In another particular embodiment, the amino acid sequenceof the LCVR is SEQ ID NO: 8, and the amino acid sequence of the HCVR isSEQ ID NO: 7. In another particular embodiment, the amino acid sequenceof the LCVR is SEQ ID NO: 12, and the amino acid sequence of the HCVR isSEQ ID NO: 11.

The present invention also provides an antibody that binds human IL-33,wherein the antibody comprises a light chain (LC) and a heavy chain(HC), and wherein the amino acid sequence of the LC is SEQ ID NO: 2, SEQID NO: 6, or SEQ ID NO: 10, and the amino acid sequence of the HC is SEQID NO: 1, SEQ ID NO: 5, or SEQ ID NO: 9. In a particular embodiment, theamino acid sequence of the LC is SEQ ID NO: 2, and the amino acidsequence of the HC is SEQ ID NO: 1. In another particular embodiment,the amino acid sequence of the LC is SEQ ID NO: 6, and the amino acidsequence of the HC is SEQ ID NO: 5. In another particular embodiment,the amino acid sequence of the LC is SEQ ID NO: 10, and the amino acidsequence of the HC is SEQ ID NO: 9.

The present invention also provides an antibody that binds human IL-33,wherein the antibody comprises 2 LCs and 2 HCs, and wherein amino acidsequence of each LC is SEQ ID NO: 2, SEQ ID NO: 6, or SEQ ID NO: 10, andthe amino acid sequence of each HC is SEQ ID NO: 1, SEQ ID NO: 5, or SEQID NO: 9. In a particular embodiment, the amino acid sequence of each LCis SEQ ID NO: 2, and the amino acid sequence of each HC is SEQ ID NO: 1.In a particular embodiment, the amino acid sequence of each LC is SEQ IDNO: 6, and the amino acid sequence of each HC is SEQ ID NO: 5. Inanother particular embodiment, the amino acid sequence of each LC is SEQID NO: 10, and the amino acid sequence of each HC is SEQ ID NO: 9.

The present invention also provides a pharmaceutical compositioncomprising an antibody of the present invention, and one or morepharmaceutically acceptable carriers, diluents, or excipients. In someembodiments, pharmaceutical compositions of the present invention can beused in the treatment of an allergic disease, whereby such treatmentcomprises administering to a patient in need thereof an effective amountof a pharmaceutical composition of the present invention. In someparticular embodiments, the allergic disease is atopic dermatitis,asthma, allergic rhinitis, or food allergy. In some embodiments,pharmaceutical compositions of the present invention can be used in thetreatment of eosinophilic esophagitis, scleroderma/systemic sclerosis,ulcerative colitis, or chronic obstructive pulmonary disease. In someembodiments, pharmaceutical compositions of the present invention can beused in the treatment of Crohn's disease.

The present invention also provides a method of treating an allergicdisease, comprising administering to a patient in need thereof aneffective amount of an antibody of the present invention. In some suchembodiments, the allergic disease is atopic dermatitis. In other suchembodiments, the allergic disease is asthma. In other such embodiments,the allergic disease is food allergy. In other such embodiments, theallergic disease is allergic rhinitis. The present invention alsoprovides a method of treating atopic dermatitis, comprisingadministering to a patient in need thereof an effective amount of anantibody of the present invention. In some embodiments, the presentinvention also provides a method of treating at least one ofeosinophilic esophagitis, scleroderma/systemic sclerosis, ulcerativecolitis, and chronic obstructive pulmonary disease, comprisingadministering to a patient in need thereof an effective amount of anantibody of the present invention. In some embodiments, the presentinvention also provides a method of treating Crohn's disease.

The present invention also provides an antibody of the present inventionor pharmaceutical composition thereof for use in therapy. In someembodiments, the present invention provides an antibody of the presentinvention or pharmaceutical composition thereof for use in the treatmentof allergic disease, wherein the allergic disease is atopic dermatitis,asthma, allergic rhinitis, or food allergy. In a particular embodiment,the present invention provides an antibody of the present invention orpharmaceutical composition thereof for use in the treatment of atopicdermatitis. In some embodiments, the present invention provides anantibody of the present invention or pharmaceutical composition thereoffor use in the treatment of eosinophilic esophagitis,scleroderma/systemic sclerosis, ulcerative colitis, or chronicobstructive pulmonary disease. In some embodiments, the presentinvention provides an antibody of the present invention orpharmaceutical composition thereof for use in the treatment of Crohn'sdisease.

In an embodiment, the present invention also provides the use of anantibody of the present invention or a pharmaceutical compositionthereof in the manufacture of a medicament for the treatment of allergicdisease. In some embodiments, the present invention provides the use ofan antibody of the present invention or a pharmaceutical compositionthereof in the manufacture of a medicament for the treatment of allergicdisease, wherein the allergic disease is atopic dermatitis, asthma,allergic rhinitis, or food allergy. In a particular embodiment, thepresent invention provides the use of an antibody of the presentinvention in the manufacture of a medicament for the treatment of atopicdermatitis. In some embodiments, the present invention provides the useof an antibody of the present invention or a pharmaceutical compositionthereof in the manufacture of a medicament for the treatment ofeosinophilic esophagitis, scleroderma/systemic sclerosis, ulcerativecolitis, or chronic obstructive pulmonary disease. In some embodiments,the present invention provides the use of an antibody of the presentinvention or a pharmaceutical composition thereof in the manufacture ofa medicament for the treatment of Crohn's disease.

The present invention also relates to the nucleic acid moleculesencoding the antibodies of the present invention. In an embodiment, thepresent invention provides a DNA molecule comprising a polynucleotidesequence encoding a HC, wherein the amino acid sequence of the HC is SEQID NO: 9. According to some such embodiments, the DNA molecule has apolynucleotide sequence given by SEQ ID NO: 20.

In an embodiment, the present invention provides a DNA moleculecomprising a polynucleotide sequence encoding a LC, wherein the aminoacid sequence of the LC is SEQ ID NO: 10. According to some suchembodiments, the DNA molecule has a polynucleotide sequence given by SEQID NO: 21.

In a further embodiment, the present invention provides a DNA moleculecomprising a polynucleotide sequence encoding a HC having the amino acidsequence of SEQ ID NO: 9, and comprising a polynucleotide sequenceencoding a LC having the amino acid sequence of SEQ ID NO: 10. In aparticular embodiment, the polynucleotide sequence encoding the HChaving the amino acid sequence of SEQ ID NO: 9 is given by SEQ ID NO:20, and the polynucleotide sequence encoding the LC having the aminoacid sequence of SEQ ID NO: 10 is given by SEQ ID NO: 21.

The present invention also provides a mammalian cell transformed withDNA molecule(s), which cell is capable of expressing a compoundcomprising a HC and a LC of the present invention, wherein the HC isgiven by SEQ ID NO: 9, and the LC is given by SEQ ID NO: 10. Also, thepresent invention provides a process for producing a compound comprisingthe HC and the LC, comprising cultivating the mammalian cell underconditions such that the antibody of the present invention is expressed.The present invention also provides an antibody produced by saidprocess.

In another embodiment, the present invention provides an antibody thatcontacts human IL-33 at a novel epitope, wherein the epitope has thefollowing residues of SEQ ID NO: 19: Ser at position 23; Pro at position24; Ile at position 25; Thr at position 26; Glu at position 27; Tyr atposition 28; Leu at position 29; Tyr at position 69; Glu at position 71;Val at position 83; Asp at position 84; Lys at position 86; Leu atposition 88; Leu at position 126; Asn at position 128; Met at position129; Asn at position 132; Cys at position 133; Val at position 134; Gluat position 175; and Thr at position 176. In such an embodiment, theepitope is determined by X-ray crystallography where any residue onIL-33 within 4.5 Å of another residue on the bound Fab is considered tobe a contact site. The term “epitope” as used herein thus refers tosites of an antigen that are in contact with the variable region of anantibody.

As used herein, an “antibody” is an immunoglobulin molecule comprising 2HCs and 2 LCs interconnected by disulfide bonds. The amino terminalportion of each LC and HC includes a variable region of about 100-120amino acids primarily responsible for antigen recognition via the CDRscontained therein. The CDRs are interspersed with regions that are moreconserved, termed framework regions (“FR”). Each LCVR and HCVR iscomposed of 3 CDRs and 4 FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. The 3 CDRs of the LC are referred to as “LCDR1, LCDR2, andLCDR3,” and the 3 CDRs of the HC are referred to as “HCDR1, HCDR2, andHCDR3.” The CDRs contain most of the residues which form specificinteractions with the antigen. That is, the CDRs contain most of theresidues that are in contact with (within 4.5 Å) the antigen's residues.The functional ability of an antibody to bind a particular antigen is,thus, largely influenced by the amino acid residues within the six CDRs.Assignment of amino acids to CDR domains within the LCVR and HCVRregions of the antibodies of the present invention is based on thewell-known Kabat numbering convention (Kabat, et al., Ann. NY Acad. Sci.190:382-93 (1971); Kabat et al., Sequences of Proteins of ImmunologicalInterest, Fifth Edition, U.S. Department of Health and Human Services,NIH Publication No. 91-3242 (1991)), North numbering convention (Northet al., A New Clustering of Antibody CDR Loop Conformations, Journal ofMolecular Biology, 406:228-256 (2011)), and Chothia (Chothia C, Lesk AM. Canonical structures for the hypervariable regions ofimmunoglobulins. J. Mol. Biol. 1987; 196:901-17. Chothia C, Lesk A M,Tramontano A, Levitt M, Smith-Gill S J, Air G, Sheriff S, Padlan E A,Davies D, Tulip W R, et al. Conformations of immunoglobulinhypervariable regions. Nature. 1989; 342:877-83). The CDRs of theantibodies of the present invention are defined according to Table 1.

TABLE 1 CDR numbering conventions used to define the CDRs of theantibodies of the present invention. Starting Amino Acid Ending AminoAcid CDR Residue Defined By: Residue Defined By: HCDR1 ChothiaKabat/North HCDR2 Kabat Kabat HCDR3 Chothia/Kabat Chothia/Kabat LCDR1Chothia/Kabat/North Chothia/Kabat/North LCDR2 Chothia/KabatChothia/Kabat/North LCDR3 Chothia/Kabat/North Chothia/Kabat/North

The antibodies of the present invention may be prepared and purifiedusing known methods. For example, cDNA sequences encoding a HC (forexample the amino acid sequence given by SEQ ID NO: 9) and a LC (forexample, the amino acid sequence given by SEQ ID NO: 10) may be clonedand engineered into a GS (glutamine synthetase) expression vector. Theengineered immunoglobulin expression vector may then be stablytransfected into CHO cells. As one skilled in the art will appreciate,mammalian expression of antibodies will result in glycosylation,typically at highly conserved N-glycosylation sites in the Fc region.Stable clones may be verified for expression of an antibody specificallybinding to IL-33. Positive clones may be expanded into serum-freeculture medium for antibody production in bioreactors. Medium, intowhich an antibody has been secreted, may be purified by conventionaltechniques. For example, the medium may be conveniently applied to aProtein A or G Sepharose FF column that has been equilibrated with acompatible buffer, such as phosphate buffered saline. The column iswashed to remove nonspecific binding components. The bound antibody iseluted, for example, by pH gradient and antibody fractions are detected,such as by SDS-PAGE, and then pooled. The antibody may be concentratedand/or sterile filtered using common techniques. Soluble aggregate andmultimers may be effectively removed by common techniques, includingsize exclusion, hydrophobic interaction, ion exchange, or hydroxyapatitechromatography. The product may be immediately frozen, for example at−70° C., or may be lyophilized.

An antibody of the present invention can be incorporated into apharmaceutical composition which can be prepared by methods well knownin the art and comprise an antibody of the present invention and one ormore pharmaceutically acceptable carriers, diluents, or excipients.

A pharmaceutical composition comprising an effective amount of anantibody of the present invention can be administered to a patient atrisk for, or exhibiting, diseases or disorders as described herein byparental routes (e.g., subcutaneous, intravenous, intraperitoneal,intramuscular, or transdermal). An “effective amount” refers to anamount necessary (at dosages and for periods of time and for the meansof administration) to achieve the desired therapeutic result. Aneffective amount of the antibody may vary according to factors such asthe disease state, age, sex, and weight of the individual, and theability of the antibody to elicit a desired response in the individual.An effective amount is also one in which any toxic or detrimentaleffects of the antibody of the present invention are outweighed by thetherapeutically beneficial effects.

The antibodies of the present invention can be used in the treatment ofpatients. More particularly the antibodies of the present invention areexpected to treat allergic diseases such as atopic dermatitis, asthma,allergic rhinitis, and food allergy. Allergic diseases are a set ofchronic conditions involving abnormal immune responses to substancesthat are ordinarily harmless to most people. The antibodies of thepresent invention are also expected to treat eosinophilic esophagitis,scleroderma/systemic sclerosis, ulcerative colitis, and chronicobstructive pulmonary disease. The antibodies of the present inventionare also expected to treat Crohn's disease.

As used interchangeably herein, “treatment” and/or “treating” and/or“treat” are intended to refer to all processes wherein there may be aslowing, interrupting, arresting, controlling, stopping, or reversing ofthe progression of the disorders described herein, but does notnecessarily indicate a total elimination of all disorder symptoms.Treatment includes administration of an antibody of the presentinvention for treatment of a disease or condition in a human that wouldbenefit from a reduction in IL-33 activity, and includes: (a) inhibitingfurther progression of the disease; and (b) relieving the disease, i.e.,causing regression of the disease or disorder or alleviating symptoms orcomplications thereof.

Antibody Engineering

A parental human anti-IL-33 antibody was optimized for binding to humanIL-33. To accomplish this, the CDRs of the isolated VH and VL wererandomized by mutagenesis and resulting antibodies screened for bindingto human IL-33 using an ELISA. Affinity enhancing mutations were thencombined to yield Antibody 23, which was then optimized using aframework library approach. For the framework library, twelve human VHframework germline genes (1-24, 1-46, 1-69, 2-5, 3-15, 3-23, 3-53, 3-72,4-04, 4-39, 5-51, and 6-01) and eight human VL framework genes (A-19,A-26, A-27, B-2, B-3, L-2, L-12, and O-2) containing Antibody 23 CDRswere synthesized and cloned into heavy and light chain human IgG4expression vectors. Following 293 HEK transient transfection of all 96heavy and light chain combinations, supernatants were assayed by ELISAfor binding to human IL-33 directly coated onto a plate and tobiotinylated IL-33 in solution following the capture of Human IgG fromsupernatants with an anti-human kappa antibody. A human antibody withCDRs derived from antibody Antibody 23, utilizing the 3-53 heavy chainhuman framework and A27 human light chain framework, was chosen forfurther development (Antibody 75). The expression of Antibody 75 intransient CHO resulted in higher expression titers as compared toAntibody 23. Furthermore, the purification of Antibody 75 resulted inhigher purification yields than Antibody 23. Reduced nonspecific bindingto Heparin was also observed with Antibody 75 as compared to Antibody23. Overall, Antibody 75 had preferred properties compared to Antibody23. Antibody 75 bound to human IL-33 with an affinity of 14.5 μM andbound to cynomolgus IL-33 with an affinity of 12,400 μM, representing an˜850-fold difference in species cross-reactivity.

Antibody 75 was then optimized for binding to cynomolgus IL-33. Toaccomplish this, the CDRs of the isolated VH and VL of Antibody 75 wererandomized by mutagenesis and resulting antibodies screened for bindingto human IL-33 and cynomolgus IL-33 by ELISA. Affinity enhancingmutations to cynomolgus which did not significantly impact affinity tohuman IL-33 were then combined to yield Antibody 54. Antibody 54 boundto human IL-33 with an affinity of 46 μM and bound to cynomolgus IL-33with an affinity of 217 μM representing a 5-fold difference in speciescross-reactivity. Antibody 54 was then further engineered to reducepotential immunogenicity, which resulted in Antibody 43. Amino acidsequence identification numbers are provided below for Antibodies 43,54, and 75.

HC LC HCVR LCVR Ab 75 SEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO: 2 NO: 3 NO:4 Ab 54 SEQ ID SEQ ID SEQ ID SEQ ID NO: 5 NO: 6 NO: 7 NO: 8 Ab 43 SEQ IDSEQ ID SEQ ID SEQ ID NO: 9 NO: 10 NO: 11 NO: 12

EXAMPLES Example 1: Expression and Purification of ExemplifiedAntibodies

The antibodies of the invention can be biosynthesized, purified, andformulated for administration by well-known methods. An appropriate hostcell, such as HEK 293 or CHO, is either transiently or stablytransfected with an expression system for secreting antibodies using apredetermined HC:LC vector ratio if two vectors are used, or a singlevector system encoding both heavy chain and light chain. Vectorssuitable for expression and secretion of antibodies from thesecommonly-used host cells are well-known.

Following expression and secretion of the antibody, the medium isclarified to remove cells and the clarified medium is purified using anyof many commonly-used techniques. For example, the medium may be appliedto a Protein A or G column that has been equilibrated with a buffer,such as phosphate buffered saline (pH 7.4). The column is washed toremove nonspecific binding components. The bound antibody is eluted, forexample, by a pH gradient (such as 0.1 M sodium phosphate buffer pH 6.8to 0.1 M sodium citrate buffer pH 2.5). Antibody fractions are detected,such as by SDS-PAGE, and then are pooled. Further purification isoptional, depending on the intended use. The antibody may beconcentrated and/or sterile filtered using common techniques. Othermaterials than the antibody, such as host cell and growth mediumcomponents, and soluble aggregates and multimers of the antibody, may beeffectively reduced or removed by common techniques, including sizeexclusion, hydrophobic interaction, cation exchange, anion exchange,affinity, or hydroxyapatite chromatography. The purity of the antibodyafter these chromatography steps is typically greater than 95%. Theproduct may be frozen at −70° C. or may be lyophilized.

Exemplified Antibody 43 was expressed either transiently in CHO cellsafter co-transfection of separate heavy chain and light chain expressionDNA vectors that incorporated the DNA sequences of SEQ ID NO: 20 and SEQID NO: 21, respectively, or was expressed stably in CHO cells aftertransfection of a single DNA vector that incorporated the DNA sequencesof both SEQ ID NO: 20 and SEQ ID NO: 21, which encode the heavy chainand light chain, respectively. Medium harvested from either a 7-daytransient CHO culture or a 14-day CHO bulk culture was clarified and theresulting crude supernatant purified by Protein A chromatography.Antibody 43 bound to Protein A resin and was eluted using low pH buffer.The eluted antibody was further purified using either preparativesize-exclusion chromatography (SEC), for material produced fromtransient CHO, or using cation exchange chromatography as a polishingstep for material produced from stable CHO. The final purity of Antibody43 was evaluated by SDS-PAGE, analytical SEC-HPLC, and LC/MS analysis.Endotoxin levels were shown to be <1 EU/mg using Endosafe-PTS analysis.Purified Antibody 43 was stored in PBS (phosphate-buffered saline), pH7.2 at 4° C.

In Vitro Binding Affinity and Kinetics.

The binding kinetics and affinity of Antibody 43 to human, cynomolgusmonkey, mouse, rat, and rabbit IL-33 is determined using a surfaceplasmon resonance assay on either a Biacore T100 or T200 instrumentprimed with HBS-EP+ (GE Healthcare, 10 mM Hepes pH7.4, 150 mM NaCl, 3 mMEDTA, 0.05% surfactant P20) running buffer and analysis temperature setat 37° C. A CM4 chip containing immobilized Protein A (generated usingstandard NHS-EDC amine coupling) on all four flow cells (Fc) is used toemploy a capture method. Antibody samples are prepared at 10 μg/mL inrunning buffer. Mouse, rat, and rabbit IL-33 samples are prepared atfinal concentrations of 1000, 500, 250, 125, 63, 31, 16, and 0 nM inrunning buffer. Human and Cynomologus IL-33 samples are prepared atfinal concentrations of 250, 125, 63, 31, 16, 8, 4, 2, 1, and 0 nM inrunning buffer. Each analysis cycle involves the steps of (1) capturingantibody samples on separate flow cells (Fc2, Fc3, or Fc4); (2)injecting 200 μL of IL-33 over all flow cells at 100 μL/min; (3)returning to buffer flow for a minimum of 10 min at 100 μL/min tomonitor complex dissociation; (4) regenerating the chip surface with twosequential injections of 7.5 microliters of glycine, pH1.5; and (5)equilibrating the chip surface for 5 minutes prior to repeating thecycle. Each IL-33 concentration is injected in duplicate. Data isprocessed using standard double-referencing and fit to a 1:1 bindingmodel using Biacore T100 Evaluation software, version 2.0.1, todetermine the association rate (on-rate, k_(on), M⁻¹ s⁻¹ units) and thedissociation rate (off-rate, k_(off), s⁻¹ units). The equilibriumdissociation constant (K_(D)) is calculated from the relationshipK_(D)=k_(off)/k_(on), and is in molar units. Three experimentalreplicates (n) are conducted for the binding of Antibody 43 to human andcynomolgus monkey IL-33.

Following procedures essentially as described above, Antibody 43 had aconcentration-dependent binding response to human IL-33 and tocynomolgus monkey IL-33. At the highest concentration of mouse, rat, andrabbit IL-33 injected (1000 nM) the binding response signal did notreach the theoretical half-maximal response signal. As a result, theK_(D) of Antibody 43 to mouse, rat, and rabbit IL-33 were estimated tobe >1000 nM. Similar methods as described above were used to determinethe binding kinetics and affinity of Antibody 75 and Antibody 54.Similar methods were also used to determine the binding kinetics andaffinity of an anti-IL-33 IgG4 antibody (not of the present invention)having the HCVR and LCVR sequences of APE4909 (disclosed in WO15106080;herein referred to as “Antibody 6”). These results are shown in Table 2.

TABLE 2 IL-33 binding kinetics and affinity of antibodies of the presentinvention and Antibody 6. k_(on) (M⁻¹s⁻¹) k_(off) (s⁻¹) K_(D) (pM)Antibody Antigen Avg ± SD Avg ± SD Avg ± SD N Antibody 75* Human IL-331.7 ± 0.5 × 10⁶ 2.6 ± 0.1 × 10⁻⁵ 14.5 ± 1.5 3 Antibody 75* Cynomolgus1.7 ± 0.6 × 10⁶ 1.9 ± 0.1 × 10⁻² 12400 ± 4000 3 IL-33 Antibody 54* HumanIL-33 1.5 ± 0.5 × 10⁶ 6.8 ± 1.3 × 10⁻⁵ 46 ± 9 3 Antibody 54* Cynomolgus1.5 ± 0.2 × 10⁶ 3.4 ± 0.5 × 10⁻⁴ 217 ± 50 3 IL-33 Antibody 43 HumanIL-33 1.5 ± 0.1 × 10⁶ 7.2 ± 1.7 × 10⁻⁵  49 ± 14 3 Antibody 43 Cynomolgus1.5 ± 0.2 × 10⁶  51 ± 2.0 × 10⁻⁵ 338 ± 45 3 IL-33 Antibody 6* HumanIL-33 1.6 ± 0.1 × 10⁶ 4.0 ± 0.9 × 10⁻⁴ 252 ± 73 2 Antibody 6* Cynomolgus1.8 ± 0.6 × 10⁶ 1.3 ± 0.1 × 10⁻³  804 ± 377 2 IL-33 ntibody 43 MouseIL-33 K_(D) > 1000 nM 1 Antibody 43 Rat IL-33 K_(D) > 1000 nM 1 Antibody43 Rabbit IL-33 K_(D) > 1000 nM 1 *Tested on different days.In Vitro Characterization of Binding to Human IL-33 and Other IL-1Family Members.

BIAcore biosensor 2000 is used to demonstrate the binding specificity ofAntibody 43 to human IL-33 and to show that the exemplified antibodydoes not bind to other members of the human IL-1 protein family.

Protein A (Calbiochem) is coupled via free amine groups to carboxylgroups on flow cells 1 and 2 of a CM5 biosensor chip (GE Healthcare)using a mixture of N-ethyl-N-(dimethylaminopropyl)-carbodiimide (EDC)and N-hydroxysuccinimide (NHS). To capture positive control antibodies,Rabbit anti-Goat IgG (Fc specific) is coupled to flow cell 3, and Rabbitanti-Rat IgG (Fc specific) is coupled to flow cell 4 (both from JacksonImmunoResearch). Flow cells are monitored with a flow rate of 30μL/minute using a buffer containing 0.01 M HEPES, pH 7.4, 150 mM NaCl,0.005% surfactant P20. Antibody 43 is captured on flow cell 2 to yield atotal of 300 to 800 response units (RU; results reflect flow cell 2minus flow cell 1). Binding tests are followed by a regeneration stepusing glycine-HCl (pH 1.5) between each cycle. Flow cell 1 is used as acontrol to monitor non-specific binding of analytes tested. Theexemplified antibody is tested with all analytes of the human IL-1protein family as listed at 500 nM concentration.

Following procedures essentially as described above, the results inTable 3 show that Antibody 43 only binds to human IL-33 and not to theother members of the IL-1 family.

TABLE 3 Antibody 43 binds specifically to IL-33. Exemplified AntibodyPositive control Ligand binding binding IL-33 Yes IL-1α No Yes IL-1β NoYes IL-1RA No Yes IL-18 No Yes IL-36α No Yes IL-36β No Yes IL-36γ No YesIL-36RA No Yes IL-37 No YesNeutralization of IL-33 in the GEC NFκB-Luciferase Reporter Assay InVitro.

Human glomerular endothelial cells (GEC) stably transfected with aNFκB-luciferase construct are used to determine the ability of Antibody43 to inhibit IL-33-induced NFκB activity. The GEC line naturallyexpresses the ST2 receptor and its co-receptor IL1RAP. In response tohuman IL-33, the NFκB pathway is activated in GECs.

GEC-NFκB-luc cells are cultured in assay medium (EGM BulletKit medium(LONZA) plus puromycin). On the day prior to the assay, GEC-NFκB-luccells are plated at 5,000 cells in 50 μl/well in white walled Collagen Itreated plates (BD Biocoat), and the plates are incubated overnight.

The next day the cells are treated with Antibody 43 in the presence ofIL-33. For each test, 25 μl of exemplified antibody is added per well ata dose range of 0 to 133.3 nM per well. 25 μl of human IL-33 is thenadded to each well at a final concentration of 126 μM (based on MW=20kDa). Human monomeric ST2 (IL1RL1) is used as a positive control in theassay in a dose range of 0 to 142.9 nM (final concentration base onMW=35 kDa), and an isotype control antibody is used as a negativecontrol in the assay in a dose range of 0 to 133.3 nM (finalconcentration base on MW=75 kDa). All samples are run in triplicate. The96-well plates are incubated at 37° C., 95% relative humidity, 5% CO₂for 4 hours, after which 100 μl/well of One-Glo Luciferase solution isadded to measure luciferase activity and plates are read on aluminometer (Perkin Elmer Victor3). The results are shown below in Table4.

TABLE 4 Exemplified antibody IC₅₀ (pM, Avg ± SD) in the in vitroNFκB-GEC-luciferase activity assay. Antibody Human IL-33 IC₅₀ Cyno-IL33IC₅₀ Antibody 43 330 ± 112 1918 ± 152 Antibody 54* 139 ± 98   851 ± 500Antibody 75* 286 ± 67  N.D. Antibody 6* 292 ± 158 1031 Positive control426 ± 115 175 ± 21 *Tested on a different day.

Following procedures essentially as described above, Antibody 43inhibits human IL-33 and cyno IL-33, but not mouse, rat, or rabbitIL-33-induced NFκB activity in a dose-dependent manner. The average IC₅₀from three independent experiments for human and cyno neutralization issummarized in Table 4. Negative control antibody did not inhibit NFκBactivity in GEC-NFκB-luc cells at any concentration tested.

Neutralization of IL-33-Induced GM-CSF Secretion from Human Mast CellsIn Vitro.

Inhibition of IL-33-induced GM-CSF release in human mast cells bytreatment with Antibody 43 is determined. Human mast cells naturallyexpressing the ST2 (IL1RL1) receptor and its co-receptor IL1RAP aredifferentiated in culture from human cord blood stem cells usingStemSpan Medium (StemCell) and SCF/IL-6. On the day of the assay, mastcells are plated at 50,000 cells in 50 μl/well in 96-well tissue cultureplates in culture medium. Cells are treated with IL-33 in the presenceor absence of antibodies. For each test, 25 μl of 4× exemplifiedantibody is added per well at dose range of 0 to 30 nM. 25 μl of 4×human IL-33 is added to each well to a final concentration 0.5 nM. Assaymedium alone is used as a no treatment control and a human monomeric ST2is used as a positive control at a dose range of 0 to 30 nM. The isotypecontrol antibody tested at 30 nM is used as a negative control. Testingis done in triplicates. The 96-well plates are placed in tissue cultureincubator (37° C., 95% relative humidity, 5% CO₂) for 16 hours. 100μl/well of supernatant are collected to measure GM-CSF levels bycommercial ELISA (R&D Systems).

Following procedures essentially as described above, Antibody 43completely inhibited human IL33-induced GM-CSF secretion from human mastcells in a dose-dependent manner, with an IC₅₀ of 0.3 nM, which wasgreater than the positive control soluble receptor IC₅₀ of 7.6 nM. Inanother similar experiment, Antibody 54 inhibited human IL33-inducedGM-CSF secretion from human mast cells with an IC₅₀ of 0.265 nM, andAntibody 6 inhibited human IL33-induced GM-CSF secretion from human mastcells with an IC₅₀ of 0.811 nM. Isotype control antibody did not inhibitIL-33 induced GM-CSF secretion. Results are representative of twoindependent experiments.

Inhibition of IL-33-Induced IL-5 Production In Vivo.

To confirm neutralization of IL-33 in vivo, to determine whetherAntibody 43 is able to neutralize human IL-33 function and inhibitproduction of mouse IL-5 in vivo, C57BL/6 mice (n=5) are injectedintraperitoneally with 0.94 mg/kg, 0.282 mg/kg or 0.094 mg/kg ofantibody or with an isotype control antibody at 0.94 mg/kg. One daypost-injection, mice are challenged with 0.025 mg/kg of human IL-33 viaintraperitoneal injection. Six hours post-human IL-33 challenge, miceare sacrificed and serum is collected. Serum is analyzed for mouse IL-5production using a commercial ELISA (R&D Systems) as per themanufacturer's instructions.

The results indicated that Antibody 43 is able to completely inhibit theproduction of mouse IL-5 in a dose dependent manner. The levels of IL-5in serum of mice injected with 0.94 mg/kg, 0.282 mg/kg or 0.094 mg/kg ofexemplified antibody were 0.03±0.004 ng/mL, 0.166±0.036 ng/mL, and0.430±0.095 ng/mL, respectively. The negative control antibody does notinhibit the human IL-33-induced production of mouse IL-5. These datademonstrate that Antibody 43 inhibits production of mouse IL-5 throughneutralization of human IL-33 in vivo.

In Vivo Efficacy of a Surrogate Mouse IL-33 Antibody in an AirwayInflammation Model

A surrogate antibody is developed to neutralize mouse IL-33 (mIL-33) foruse in preclinical disease models. The surrogate is a murine IgG1monoclonal antibody that neutralizes mIL-33 in in vitro assays. Theability of systemic administration of the anti-mIL-33 antibody to affectthe airway inflammatory response to Alternaria challenge is determinedin vivo. Female BALB/c mice (n=5 per group) are injected subcutaneouslywith 25 mg/kg of anti-mIL-33 or isotype control antibody on Day 0. MouseST2-Fc, a soluble form of one of the co-receptors for IL-33 that canneutralize mIL-33 in in vitro assays (Positive control), is injected at12.5 mg/kg intraperitoneally on Days 1 and 2, 30 minutes prior toAlternaria administration. Alternaria extract (50 μg in 20 μL PBS) isadministered intra-nasally to each mouse on days 1 and 2. Mice aresacrificed on day 3 with CO₂ inhalation and blood is immediatelycollected via cardiac puncture for preparation of serum.

Bronchial alveolar lavage fluid (BAL) is prepared by lavaging lungs with10 washes (500 μL each wash) of PBS injection and withdrawal through acannula in the exposed trachea of each mouse. BAL fluid is centrifugedat 200 g for 10 minutes and supernatant is removed and frozen. Cells areresuspended in 1 mL ACK buffer (Sigma) to lyse the red blood cells,washed and are resuspended in 0.5 mL of PBS for counting. Eosinophilsare counted using eosinophil diluting fluid and stain (ENG Scientific,Cat. ES-3101) and total cells are counted with trypan blue. BAL fluid isassayed using commercial mouse IL-5 and Chitinase 3-like 3/YM1(Chi3L3/YM1) ELISAs (R&D Systems) and for antibody exposure. The resultsare shown below in Table 5.

TABLE 5 Levels of IL-5 and Chi3L3/YM1, and number of eosinophils andtotal number of cells in BAL fluid. Isotype Anti-mIL- PositiveCells/Cytokines Unchallenged Control 33 Control Eosinophils 32000 ±340000 ± 120000 ± 112000 ± (# cells) 13565 70711 51769 57480 IL-5(pg/mL) 2 ± 69.6 ± 2 ± 9.2 ± 0 27.31 0 6.248 Chi3L3/YM1 17.4 ± 108.6 ±27.4 ± 37.4 ± (ng/mL) 2.064 28.7 4.986 6.313 Total Cells 104000 ± 408000± 144000 ± 152000 ± (# cells) 20396 95833 31241 44989

Following procedures essentially as described above, Alternariachallenge increased the number of cells, mostly eosinophils, present inthe BAL fluid. It also induced the production of cytokines such as IL-5and Chi3L3/YM1. Systemic administration of the anti-mIL-33 antibodysignificantly reduced eosinophil and total cell infiltration as measuredby the BAL cell counts, and decreased IL-5 and Chi3L3/YM1 levels in theBAL fluid. These results demonstrate that inhibition of mIL-33 reducedmultiple inflammatory markers in this airway inflammation model.

Epitope Mapping by X-Ray Crystallography

X-Ray Crystallography

A 10.6 mg/ml solution of hIL33:FAB complex is screened for crystalsutilizing custom and commercially available screens. Vapor diffusionexperiments are set up at 22° C. as 400 nL+400 nL (protein+wellsolution) crystallization drops with 50 μL well solution (0.2 M AmmoniumSulfate and 30% w/v Polyethylene Glycol 8,000) in a MRC2 (SWISSCI)sitting drop plate. 20 mM Magnesium Acetate is added to the protein toaid in crystallization. Resulting crystals are flash frozen in liquidnitrogen following a quick dip in well solution containing 20% EthyleneGlycol added as a cryogenic agent. X-ray diffraction data are collectedusing synchrotron radiation at LRL-CAT 31-ID (Advanced Photon Source,Argonne, Ill.). 180 frames are collected at 1° oscillation at awavelength of 0.97931 Å and processed with CCP41 packages.

The crystals are of the P21212 space group with unit cell dimensionsa=70.1 Å b=194.9 Å c=46.5 Å α=β=γ=90°. A molecular replacement solutionof the hIL33:FAB complex is obtained with Phaser2 using input modelsfrom PDB structure 4KC3 and a FAB model generated with the MOE3(v2014.9) antibody modeling tool. The molecular replacement solutioncontains 1 complex in the asymmetric unit. This solution subsequentlyundergoes multiple rounds of refinement and model building with Buster4and Coot5 yielding a final structure with Rwork of 17.8% and Rfree of20.3% at 1.40 Å.

The hIL33 epitope is mapped for the high resolution crystal structure ofhIL33:FAB complex using the Protein Contacts tool in MOE3 (v2015.10).The tool is used to evaluate “distance”, “covalent”, “arene”, “ionic”,and “hbond” interactions. The output for the interchain interactions isdistilled down to the residue level in Microsoft Excel to identify thehIL33 epitope residues. The final list contains hIL33 residues that arewithin 4.5 Å of any FAB residue.

Following procedure essentially as described above, an antibody havingthe same CDRs as Antibody 75 contacts human IL-33 at an epitope given bythe following residues of SEQ ID NO: 19: Ser at position 23; Pro atposition 24; Ile at position 25; Thr at position 26; Glu at position 27;Tyr at position 28; Leu at position 29; Tyr at position 69; Glu atposition 71; Val at position 83; Asp at position 84; Lys at position 86;Leu at position 88; Leu at position 126; Asn at position 128; Met atposition 129; Asn at position 132; Cys at position 133; Val at position134; Glu at position 175; and Thr at position 176. Antibody 54 andAntibody 43 also contact a substantially similar epitope on human IL-33.

Sequences  HC of Antibody 75 (SEQ ID NO: 1)EVQLVETGGGLIQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTLHGIRAAYDAFIIWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG LC of Antibody 75 (SEQ ID NO: 2)EIVLTQSPGTLSLSPGERATLSCRASQSVGINLSWYQQKPGQAPRLLIYGASHRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCHQYSQSPPFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC HCVR of Antibody 75 (SEQ ID NO: 3)EVQLVETGGGLIQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTLHGIRAAYDAFIIWGQGTLVTVSS  LCVR of Antibody 75 (SEQ ID NO: 4)EIVLTQSPGTLSLSPGERATLSCRASQSVGINLSWYQQKPGQAPRLLIYGASHRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCHQYSQSPPFTFG GGTKVEIK HC of Antibody 54 (SEQ ID NO: 5)EVQLVETGGGLIQPGGSLRLSCAASGFTFSFYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTLHGIRAAYDAFIIWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG LC of Antibody 54 (SEQ ID NO: 6)EIVLTQSPGTLSLSPGERATLSCRASQSVGINLSWYQQKPGQAPRLLIYGASHRLTGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCHQYSQPPPFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGECHCVR of Antibody 54 (SEQ ID NO: 7)EVQLVETGGGLIQPGGSLRLSCAASGFTFSFYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTLHGIRAAYDAFIIWGQGTLVTVSS LCVR of Antibody 54 (SEQ ID NO: 8)EIVLTQSPGTLSLSPGERATLSCRASQSVGINLSWYQQKPGQAPRLLIYGASHRLTGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCHQYSQPPPFTFG GGTKVEIKHC of Antibody 43 (SEQ ID NO: 9)EVQLVETGGGLIQPGGSLRLSCAASGFTFSFYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTIEGIRAAYDAFIIWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGLC of Antibody 43 (SEQ ID NO: 10)EIVLTQSPGTLSLSPGERATLSCRASQSVGINLSWYQQKPGQAPRLLIYGASHRLTGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCHQYSQPPPFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGECHCVR of Antibody 43 (SEQ ID NO: 11)EVQLVETGGGLIQPGGSLRLSCAASGFTFSFYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTIFIGIRAAYDAFIIWGQGTLVTVSS  LCVR of Antibody 43 (SEQ ID NO: 12)EIVLTQSPGTLSLSPGERATLSCRASQSVGINLSWYQQKPGQAPRLLIYGASHRLTGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCHQYSQPPPFTFG GGTKVEIK HCDR1 (SEQ ID NO: 13)  GFTFSXYAMS Wherein X at position 6 is S or F.HCDR2 (SEQ ID NO: 14) AISGSGGSTYYADSVKG HCDR3 (SEQ ID NO: 15)TXHGIRAAYDAFII Wherein X at position 2 is L or I. LCDR1 (SEQ ID NO: 16)RASQSVGINLS LCDR2 (SEQ ID NO: 17) GASHRXTWherein X at position 6 is A or L. LCDR3 (SEQ ID NO: 18) HQYSQXPPFTWherein X at position 6 is S or P.Human IL-33 Amino Acids 95-270 (SEQ ID NO: 19)AFGISGVQKYTRALHDSSITGISPITEYLASLSTYNDQSITFALEDESYEIYVEDLKKDEKKDKVLLSYYESQHPSNESGDGVDGKMLMVTLSPTKDFWLHANNKEHSVELHKCEKPLPDQAFFVLHNMHSNCVSFECKTDPGVFIGVKDNHLALIKVDSSENLCTENILFKLSET DNA Encoding the HC of SEQ ID NO: 9 (SEQ IDNO: 20) GAGGTGCAGCTGGTGGAGACTGGAGGAGGCTTGATCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCTTTTATGCTATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGATTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTTCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAACGATCCACGGTATACGCGCAGCCTATGATGCTTTTATTATCTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAGCTTCTACCAAGGGCCCATCGGTCTTCCCGCTAGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCACCCTGCCCAGCACCTGAGGCCGCCGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAG CCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAAAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTDNA Encoding the LC of SEQ ID NO: 10 (SEQ ID NO: 21)GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTGGCATCAACTTGTCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCCATAGGCTAACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCATCAATATAGTCAACCACCTCCCTTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGGACCGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGC

We claim:
 1. An antibody that binds human IL-33, comprising a lightchain variable region (LCVR) and a heavy chain variable region (HCVR),wherein the LCVR comprises complementarity determining regions (CDRs)LCDR1, LCDR2, and LCDR3 and the HCVR comprises CDRs HCDR1, HCDR2, andHCDR3, and wherein the amino acid sequence of LCDR1 is SEQ ID NO: 16,the amino acid sequence of LCDR2 is SEQ ID NO: 17, the amino acidsequence of LCDR3 is SEQ ID NO: 18, the amino acid sequence of HCDR1 isSEQ ID NO: 13, the amino acid sequence of HCDR2 is SEQ ID NO: 14, andthe amino acid sequence of HCDR3 is SEQ ID NO: 15, and wherein Xaa atposition 6 of SEQ ID NO: 13 is Ser or Phe, Xaa at position 2 of SEQ IDNO: 15 is Leu or Ile, Xaa at position 6 of SEQ ID NO: 17 is Ala or Leu,and Xaa at position 6 of SEQ ID NO: 18 is Ser or Pro.
 2. The antibody ofclaim 1, wherein: a. Xaa at position 6 of SEQ ID NO: 13 is Ser; b. Xaaat position 2 of SEQ ID NO: 15 is Leu; c. Xaa at position 6 of SEQ IDNO: 17 is Ala; and d. Xaa at position 6 of SEQ ID NO: 18 is Ser.
 3. Theantibody of claim 1, wherein: a. Xaa at position 6 of SEQ ID NO: 13 isPhe; b. Xaa at position 2 of SEQ ID NO: 15 is Leu; c. Xaa at position 6of SEQ ID NO: 17 is Leu; and d. Xaa at position 6 of SEQ ID NO: 18 isPro.
 4. The antibody of claim 1, wherein: a. Xaa at position 6 of SEQ IDNO: 13 is Phe; b. Xaa at position 2 of SEQ ID NO: 15 is Ile; c. Xaa atposition 6 of SEQ ID NO: 17 is Leu; and d. Xaa at position 6 of SEQ IDNO: 18 is Pro.
 5. The antibody of claim 1, wherein the amino acidsequence of the LCVR is SEQ ID NO: 4, SEQ ID NO: 8, or SEQ ID NO: 12,and the amino acid sequence of the HCVR is SEQ ID NO: 3, SEQ ID NO: 7,or SEQ ID NO:
 11. 6. The antibody of claim 5, wherein the amino acidsequence of the LCVR is SEQ ID NO: 4, and the amino acid sequence of theHCVR is SEQ ID NO:
 3. 7. The antibody of claim 5, wherein the amino acidsequence of the LCVR is SEQ ID NO: 8, and the amino acid sequence of theHCVR is SEQ ID NO:
 7. 8. The antibody of claim 5, wherein the amino acidsequence of the LCVR is SEQ ID NO: 12, and the amino acid sequence ofthe HCVR is SEQ ID NO:
 11. 9. The antibody of claim 1, wherein theantibody comprises a light chain (LC) and a heavy chain (HC), andwherein the amino acid sequence of the LC is SEQ ID NO: 2, SEQ ID NO: 6,or SEQ ID NO: 10, and the amino acid sequence of the HC is SEQ ID NO: 1,SEQ ID NO: 5, or SEQ ID NO:
 9. 10. The antibody of claim 9, wherein theamino acid sequence of the LC is SEQ ID NO: 2, and the amino acidsequence of the HC is SEQ ID NO:
 1. 11. The antibody of claim 9, whereinthe amino acid sequence of the LC is SEQ ID NO: 6, and the amino acidsequence of the HC is SEQ ID NO:
 5. 12. The antibody of claim 9, whereinthe amino acid sequence of the LC is SEQ ID NO: 10, and the amino acidsequence of the HC is SEQ ID NO:
 9. 13. A pharmaceutical compositioncomprising the antibody of claim 1, and one or more pharmaceuticallyacceptable carriers, diluents, or excipients.
 14. A method of treatingone or more allergic disease, comprising administering to a patient inneed thereof an effective amount of the antibody of claim
 1. 15. Themethod of claim 14, wherein the allergic disease is atopic dermatitis,asthma, allergic rhinitis, or food allergy.
 16. A method of treatingatopic dermatitis, comprising administering to a patient in need thereofan effective amount of the antibody of claim
 1. 17. A method of treatingat least one of eosinophilic esophagitis, scleroderma/systemicsclerosis, ulcerative colitis, and chronic obstructive pulmonarydisease, comprising administering to a patient in need thereof aneffective amount of the antibody of claim 1.